The invention relates to a microscope according to the preamble of claim 1. This is essentially a novel type of illuminating system for microscopy. The advantages and application possibilities of the invention for lighting of any type in the field of microscopy are plain to the person skilled in the art. However, in a way that is representative of the most varied types of illumination, consideration is given below, in particular, to the distinguishing features in the field of illumination by means of fluorescent light for which the invention is preferably to be put to use. Although the invention is advantageous for the most varied types of microscopes, stereomicroscopes will be considered below in particular.
Ploem""s reflected light principle is frequently employed for fluorescence microscopy. The discovery of the green fluorescing protein GFP (Green Fluorescent Protein) has produced a need for fluorescence stereo microscopy, because the non-toxic GFP can also be used to investigate living organisms. Thanks to the upright, non-reversed and three-dimensional image and to the large working distance, the stereomicroscope is better suited for this purpose than the classic 2D microscope. The latter is represented, for example, in the Leica company document 913894. The following solutions are known for fluorescence microscopy using 3D microscopes:
a) Separate Illumination:
The observer uses a conventional stereomicroscope. A blocking filter is inserted into the observation beam paths at a suitable point. This can be a filter which covers both beam paths, or one filter each in the two beam paths of the stereomicroscope. The illumination of the object is performed independentlyxe2x80x94for example to the sidexe2x80x94of the stereomicroscope optical system. The exciter filter is arranged in the illuminating beam path. Such a design is to be found, for example, in DE-A-2348567, an annular light tube being applied there which surrounds the main objective and, for its part, is shielded from the surroundings by a bellows. A transmitted-light microscope with fluorescent illumination toward the objective has, for example, been marketed under the designation xe2x80x9cLeitz Fluoreszenz Mikroskop mit Orthomatxe2x80x9d [xe2x80x9cLight Fluorescence Microscope with Orthomatxe2x80x9d].
b) Illumination by the Stereomicroscope:
A Ploem fluorescent illuminator having an exciter filter, a dichromatic beam-splitting mirror and blocking filter is arranged in each beam path between the magnification power changer and the binocular tube of the stereomicroscope. It suffices if the illumination is coupled only into one of the two beam paths. There is then no need for the exciter filter in the other beam path. The dichromatic beam-splitting mirror and/or the blocking filters can be combined to form one component which covers both beam paths. Such a system is described, for example, in the company documents: xe2x80x9cLeica Stereo-Fluoreszenzsystemxe2x80x9d [Leica Stereo Fluorescence Systemxe2x80x9d] (printed publication No.: M1-203-3de; IV.96) and xe2x80x9cLeica-Stereo-Fluoreszenzsystemxe2x80x9d [xe2x80x9cLeica Stereo Fluorescence Systemxe2x80x9d] (printed publication No.: M1-203-4de; IV.96), and is likewise represented in an earlier Leica company document (printed publication No.: M1-143-4de; II.97).
Units for non-fluorescing illumination through the microscope are also represented, for example, in documents U.S. Pat. No. 3,512,860, DE-A-3 339 172 and DE-A-3 427 592. In the design in accordance with the first and second citations, light is reflected directly into the observation beam path via beam-splitting mirrors upstream of or through the main objective; in the case of the design in accordance with the third citation, an illuminating beam path is directed onto the object next to the main objective obliquely relative to the observation beam path. Comparable to the second citation is an illuminating system for the applicant""s xe2x80x9cM650xe2x80x9d stereomicroscope in which the illuminating beam path additionally also has a magnification power changer which is mechanically coupled to the magnification power changer of the observation beam paths, with the result that both the image field illumination and the brightness are adapted to the respective magnification power, as represented, for example, in the Leica company document with the printed publication no.: M1-601-0fr; IX.94.
This prior art has the following disadvantages:
a) In the Case of Separate Illumination:
In the event of a change in the magnification power, the illuminating optical system is not adapted to the changing size of the object field. Consequently, in the case of weak magnification only a portion of the object field is illuminated and/or in the case of high magnification invisible object paths are also needlessly illuminated. Consequently, the illumination level is lower in the case of a high magnification power than if the entire luminous flux were concentrated onto the small object field.
b) In the Case of Illumination Through the Stereomicroscope:
In the magnification path changer, the exciting light floods the same optical components which are also used to observe the fluorescing object. This requires all these optical components to have a high transparency to UV and blue light, and to be free from autofluorescence. Depending on the selected magnification power, it is also possible for parts in the surrounding field of the lenses also to be undesirably irradiated with exciting light. There, as well, no fluorescent light may be generated. In practice, these requirements can be met only with a high outlay. To date, compromises have been made in equipment design: thus, on the one hand, the illumination level of the object with exciting light is reduced and, on the other hand, undesired fluorescent light which is superimposed on that from the object and thus impairs the image quality is generated in the magnification power changer or in the observation beam path or paths. Moreover, autofluorescence occurring from lens cement, dust and housing parts may cause additional disturbance.
The abovementioned microscopes of the applicant (for example xe2x80x9cM650xe2x80x9d, or else xe2x80x9cM690xe2x80x9dxe2x80x94with a zoomxe2x80x94), which have a dedicated illuminating beam path with setting of the magnification power, the magnification power changer of the observation beam paths being coupled to the magnification power changer of the illumination beam path such that the object illumination is basically adapted to the respective magnification power, require precisely a spatially separated, additional magnification power changer and a corresponding linkage, for which reason these fittings are very bulky. The application of such a microscope through the replacement of the conventional illumination by a fluorescent light source and appropriate filters has moreover, also not yet been suggested. The point is that the problem of the autoreflection of undesired fluorescent light beams between the main objective and the magnification power changer may also occur in such a case; the reason for this, in particular, is that the illuminating beam path reflected in strikes the main objective at a certain angle which is oblique to the observation beam paths.
It is therefore the aim of the present invention to create a microscope having an optimized illuminating beam path, in particular to create a microscope, preferably a stereomicroscope, for observing fluorescing objects which avoids the disadvantages of the state of the art. In particular, the aim is to adapt the object illumination with exciting light to the magnification power respectively selected. In addition, the illuminating optical system is to be highly transparent to UV light and blue light, and the image quality may not be impaired by autofluorescence internal to the instrument.
It is certainly already regarded as inventive [lacuna] one inventive idea for the purpose of converting a microscope resembling the xe2x80x9cM650xe2x80x9d or xe2x80x9cM690xe2x80x9d of the applicant into a fluorescence microscope by replacing the conventional light source with a fluorescent light source and installing appropriate exciting and blocking filters. Such a novel design would still have the advantage that the exciting light falls on the object in a concentrated fashion which is thus economical in applicationxe2x80x94specifically independently of the respectively selected magnification power of the observation beam paths of these microscopes. However, the invention goes beyond this and also attempts to improve such designs further.
Furthermore, it is proposed according to the invention to supplement the magnification power changer of the stereomicroscope with a third beam path for the exciting light. The two observation beam paths are therefore no longer flooded with the exciting light. Any possible autofluorescence in the illuminating beam path does not disturb the observation. The illuminating beam path can be optimized to provide an improved transparency to UV and blue light, without having to pay regard to the imaging quality of the observation beam path. The exciting light is coupled into the abovementioned third beam path of the magnification power changer in a very simple way; no dichromatic beam-splitting mirror is required for this purpose. The changing device for the exciting filter and the blocking filter can thus be of simpler configuration.
Especially when the coupling-in is performed such that all the beam paths run parallel in the region of the magnification power changer, it is also impossible for disturbing autoreflections to occur on the inside of the main objective, since these would, if appropriate, be retroreflected into the illuminating beam path.
Further advantageous refinements and variants thereon are described in the patent claims and are protected. Additional variants follow from the description of the figures.
The stereomicroscope in accordance with the present invention can be applied both in Greenough stereomicroscopes and in stereomicroscopes having a common main objective.
If a UV safety screen (for example xe2x80x9cGG 420xe2x80x9d) is additionally permanently installed in the observation beam path, the observer is protected against harmful UV light even when the blocking filter is removed.
If the exciting filter and the blocking filter are removed, the object can be observed in the reflected light. Since the illumination strikes the object very steeply, this device is ideal for investigating deep holes. The replacement or the additional arrangement of a normal light source for the illuminating beam path is easy to solve in technical terms. For example, it would also be possible to provide a changeover switch or a capping plate which introduce permanently installed normal light into the illuminating beam path via a beam-splitting mirror, as has been implemented, for example, with the known xe2x80x9cLN Galileoxe2x80x9d transmitted-light fluorescence microscope.
It is advantageous for the exciting filter and the blocking filter to be changeable independently of one another, in order to be able to provide any desired filter combinations. According to the invention, in this case the filters are mounted on a filter changer, designed as a disk or slide, to form a plurality of sets of exciting and blocking filters. It is possible thereby to switch over quickly between various fluorescence methods. A shutter which is preferably arranged and is driven by the filter disk interrupts the illuminating beam path when a change is made from one filter set to the other. This ensures the safety of the viewer. A slide which is possibly additionally present permits the illuminating beam path also to be interrupted manually. In this way, the microscope can also be used with conventional illumination without disconnecting the light source for fluorescence viewing. This advantageously eliminates waiting during cooling and restarting of the fluorescence lamp.
The filter sets can be operated effectively by being embedded in filter carriers, and are protected against contamination and damage by being held in the filter changer. By contrast with expensive and bulky dichroic mirrors or prisms which have been applied in known instruments as exchangeable filters for reflection into the observation beam path, the flat filter sets according to the invention are more cost-effective and can be installed integrally in an optimum fashion.
The filters are changed without aids, resulting in optimization of the operating speed.
As is known per se, it is possible to connect in parallel with the observation beam path at least one assistant""s tube whose beam path is reflected out via at least one partial reflecting surface between the eyepieces of the observation beam path and the filters. Assistants can thereby view the fluorescing object without difficulty.
In order to optimize space (all beam paths in a cylindrical space which is as narrow as possible), in accordance with a particular refinement of the invention the center of the main objective was displaced eccentrically relative to the cross section of this cylindrical space.
The illuminating beam path can also be split up for the purpose of further optimizing space, in which case the axes of the observation beam paths are once again displaced in a radial plane of the cylindrical space. The cylinder diameter can be further reduced in this design.
Alternatively, all the beam paths can jointly penetrate a main objective, or each beam path per se can have a dedicated main objective, which would entirely exclude any disturbing reflections possibly occurring.
If oblique illumination of the object is desired for technical reasons concerning illumination, it is also possible to provide downstream of the main objective an appropriate optical element which directs the illumination appropriately.
To protect the observers and those not involved, it is conceivable to provide concentrically with the main objective a bellows which shields the space between the object and main objective from the outside.
The invention is explained below with reference to examples which have been realized in practice but which are not restrictive. Since one field of application is stereo fluorescence microscopy, it will be demonstrated in particular with reference to a novel exemplary stereo fluorescence microscope. However, it is in no way limited to fluorescence microscopy, but also decidedly comprises all other types of optical microscopy, as will become unequivocally clear to a person skilled in the art upon study of the patent claims. Since the arrangement of the new illuminating beam path is essentially identical in these other types of optical microscopy to the case of fluorescence microscopy, for the person skilled in the art the following description also results in the best method of embodying the invention for these other types, taking account of the respective, known characteristic features of these other types. Thus, for example, in most other types the installation of fluorescence filters or the like is eliminated.
The figures are described inclusively. Identical reference symbols signify identical components; reference symbols with different reference numerals signify components which have the same function but differ in design or position.